The mammalian sperm acrosome is a membrane bounded organelle which contains a variety of hydrolases that are utilized to penetrate the egg investments. The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular composition which perform specific functions required for mammalian fertilization. Our understanding of both the assembly and functions of the outer acrosomal membrane-matrix complex (OMC) polypeptides is limited. The long range goals of this proposal are to identify the molecular and structural mechanisms involved in the assembly of the outer acrosomal membrane (OAM) domain, and its adherent acrosomal matrix, and to define the role of their constituent polypeptides in the membrane fusion, and the release of hydrolases during the acrosome reaction. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38-19kDa) polypeptides. Recently, we published the purification a 45kDa polypeptide (OMC45) from the high pH insoluble fraction of OMC by continuous elution SDS-PAGE. However, the molecular characterization of OMC45 polypeptide is not known. Three aims will address these goals. Aim#1- Experiment #1a: Biochemical and Proteomic Identification of OMC45 Polypeptide: The OMC45 polypeptide sequence data derived from proteomic analyses will be used for synthesis of 20-mer peptides corresponding to the N- and C-termini of OMC45. These will be used for the acrosome reaction competitive inhibition assay in experiment #1b. Experiment #1b: To Define the Function of OMC32 (32kDa polypeptide isolated from high pH soluble fraction of OMC) and OMC45 Polypeptides Using a Permeabilized Sperm Model for Analysis of the Acrosome Reaction: This study will determine the role of OMC32 and OMC45 polypetides in the calcium-dependent membrane fusion events of acrosomal exocytosis. Experiment #1c: To identify the Interaction of the known Members of the OMC Complex by Cross-Linking Studies: This experiment will reveal the interaction of the known members of the OMC complex. Aim#2- Identification of Proteins which Mediate the Adhesion Between the OAM and the Acrosomal Matrix. The acrosomal matrix and the acrosomal membrane of mature spermatozoa are each segregated into domains of unique structure, composition and function. We found that different elements of hamster sperm acrosomal matrix are precisely localized and specifically associated with the fusigenic domains of OAM suggesting specific adhesive interactions between OAM and matrix. We hypothesize that in addition to its fusigenic process function in the acrosome reaction, the outer acrosomal membrane and matrix polypeptides interact in a receptor-ligand type of interaction to establish the outer acrosomal membrane-matrix complex and to regulate the assembly and distribution of distinct acrosomal matrix elements. Affinity chromatography and immunoprecipitation will be utilized to identify protein(s) responsible for membrane-matrix adhesion. Aim#3- To Identify the Intramolecular Post-translational Modifications of Acrosomal Matrix Polypeptides during Epididymal Transit: Our hypothesis is that the post- translational modifications of acrosomal matrix proteins occur during maturation and these biochemical modifications may be involved in the interaction of acrosomal hydrolases with matrix polypeptides. Processing of the polypeptides will be identified by 2-D PAGE and immunoblot analyses. Completion of these studies will provide new insights into the assembly of the acrosomal segment and of its functions during mammalian fertilization. Potential practical benefits derived from this work may include the development of strategies for maintaining acrosomal integrity in stored spermatozoa, promoting the acrosome reaction, and improving the fertilizing capacity of functionally impaired spermatozoa.